Clostridium perfringens type B, C and D isolates have significant medical, veterinary and biodefense importance. Many highly lethal toxins (such as beta toxin and epsilon toxin, a class B select toxin) produced by type B-D isolates are encoded by large plasmids. The long-term goal of this project is to understand the contributions of these large toxin-encoding plasmids and their encoded toxins to the pathogenicity of type B-D isolates in order to improve the design of vaccines/therapeutics against natural or bioterrorism-related human or animal infections. This work will also lead to development of subtyping assays for molecular epidemiologic or forensic investigations of natural or bioterrorism disease outbreaks involving type B-D isolates. It also has significant implications for understanding the virulence evolution of the major clostridial enteropathogens. To accomplish these goals, the following specific aims will be pursued, i) to determine which known toxins contribute to the pathogenicity of type B-D isolates, Aim A will continue constructing single and multiple toxin null mutants in type B-D backgrounds, using our recently-developed, highly efficient intron mutagenesis approaches; ii) Aim B will compare the pathogenicity of those toxin mutants versus their parent type B-D isolates using our recently optimized animal models that evaluate specific disease aspects, including enteric pathogenicity (using rabbit or goat ileal loops) or lethality (using mouse i.d. or gastric challenge models); when mutants show attenuated pathogenicity, they will be complemented to confirm the attenuation specifically resulted from inactivation of the implicated toxin gene, iii) since we have shown the epsilon toxin-encoding plasmid of type D isolates is conjugative, Aim C will examine whether the toxin plasmids of type B, C and E isolates are also conjugative (using mixed mating approaches), whether type B-D conjugative toxin plasmid transfer can occur in the intestines where this transfer may contribute to pathogenesis, whether C. perfringens can conjugatively exchange toxin plasmids or toxin genes with Clostridium difficile, another major clostridial enteropathogen, and whether certain C. perfringens toxin plasmids are incompatible with one another; and, finally, iv) Aim D will evaluate the genotypic diversity of toxin plasmids in type B and C isolates using pulsed- field gel/Southern blot and plasmid diversity in type B-D isolates using microarray approaches.